The long term goal of the proposed studies is to define the microscopic mechanisms by which water moves across biological membranes and the regulatory mechanisms by which vasopressin-sensitive epithelia exert direct control over transepithelial water permeability. This proposal is a direct extension of work performed over the past 3 year grant period. Research is focused on mammalian renal epithelia: the proximal tubule, containing fixed water channels, and the collecting tubule, containing vasopressin- inducible water channels. The three major goals for the next grant period are: 1) To continue the physiological and biochemical characterization of water channels in proximal tubule apical and basolateral membranes. Optical and NMR methods will be developed to measure osmotic and diffusional water transport and salt reflection coefficients in isolated membrane vesicles. Group-specific reagents will be tested for water transport inhibition. A liposome reconstitution assay will be developed and used to screen membrane proteins for water transport activity. 2) To examine the mechanisms of regulation of collecting tubule water permeability by vasopressin. Fluorescence microscopy methods will be developed to measure the time course of osmotic and diffusional water permeability, cell calcium, membrane fluidity and exo-/endocytosis in response to effectors of the hydroosmotic response. The pre-steady-state kinetics of vasopressin action will be studied to address fundamental questions about the nature and sequence of events by which water transport turns on and off in response to vasopressin. 3) To characterize and purify the vasopressin-sensitive water channel using isolated membranes from collecting tubule. A strategy will be developed to isolate and purify vasopressin-induced endosomes containing water channels. The biochemical and transport properties of the vasopressin-sensitive water channel will be established using the endosomes. Membrane proteins will be reconstituted into liposomes for functional analysis.